a. Exclusion of APOBEC from viral particles by HTLV-1 and SRV-1 Gag proteins. Different retroviruses have developed different strategies to avoid restriction by the APOBEC3 (A3) proteins. We have found that HTLV-1 excludes many A3 proteins from the virus particle, while SRV-1 efficiently prevents packaging of rhesus A3G and A3F but is more sensitive to A3 proteins from other species. In HTLV-1 the ability to exclude A3 mapped to NC but we have not determined the mechanism of the exclusion. In contrast, SRV-1 NC is not involved in the more species specific prevention of A3 packaging. b. Qualitative different packaging of A3B. The nuclear A3B protein has been reported to have intermediate restriction activity against HIV and to not be targeted by Vif. We found A3B to be inactive in our HIV reporter vector system but saw strong restriction when A3B was redirected to the cytoplasm [by replacing the first 60 amino acids in A3B with those of A3G (A3GB)]. This was not a consequence of different levels of incorporation into virus particles, as all A3B was efficiently packaged, and present in viral cores. However, A3GB causes 6-fold higher levels of hypermutation than A3B. We are doing additional experiments to understand these differences. c. Primate-lineage Apobec3 gene evolution. The A3 locus of old world primates (OWP) consists of seven genes made up by monomer or dimers of three paralogous subunits termed Z1, Z2, and Z3. The only other organism in the same superclade of mammals for which we have information on the A3 locus is the mouse, which has only one gene with a Z2-Z3 configuration. To expand our knowledge about the evolution of the primate A3 genes, we studied the A3 genes of new world primates (NWP), and we are in the final stages of assembling the sequence of the A3 locus of squirrel monkeys and comparing it to a recent preliminary assembly from the marmoset genome project. We found that the overall configuration of the NWP A3 locus is similar but distinct from that of OWP. NWP also have multiple loci encoding Z1, Z2 and Z3 with a Z1 type gene at the 5 and a Z3 gene at the 3 end. In addition they have at least two A3G paralogues but are missing an A3B gene. The fact that all primates have a syntenic A3G-derived processed pseudogene indicates that this gene arose over 40 million years ago. We have not yet succeeded in determining whether this gene exists in prosimians. To assess the antiviral activity of the NWP A3 genes we are constructing expression clones to compare their activity to that of old world primates, using a panel of retroviruses including squirrel monkey retrovirus.